Journal: Journal of Cellular and Molecular Medicine

Article Title: Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer

doi: 10.1111/jcmm.18033

Figure Lengend Snippet: AIL with OLP blocks HSP90‐BRCA1 and PARP1 activity. The protein expression of HSP90, BRCA1, PARP1 in two GC cell lines after treated with a blank control group, OLP, AIL and OLP + AIL group. (B) The protein expression of HSP90, BRCA1 in two GC cell lines after being treated with a specified concentration of AIL for 24 h. (C) Pull‐down of selected proteins with HSP90 in the nucleus and cytoplasm of GC cells treated with AIL for 24 h, and then the IP fractions were immunoblotted with HSP90, BRCA1 and GAPDH. (D) Representative images of HSP90, BRCA1 and PARP1 in PDX tumour tissue as detected via immunohistochemistry. Scale bars, 200 μm. (E) Data were derived from experiments conducted in triplicate in (D). The PDX tumour tissues were compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: BRCA1 (1:100, Proteintech, 22362‐1‐AP).

Techniques: Activity Assay, Expressing, Control, Concentration Assay, Immunohistochemistry, Derivative Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ailanthone synergizes with PARP1 inhibitor in tumour growth inhibition through crosstalk of DNA repair pathways in gastric cancer

doi: 10.1111/jcmm.18033

Figure Lengend Snippet: AIL inhibits BRCA1 through downregulating P23 and inevitably suppress the HR pathway of GC. Western blotting was performed to detect the expression of P23, HSP90 and BRCA1 in AGS and SGC7901 cells after treatment with a specified concentration of CEL (P23 inhibitor) for 24 h. (B) The mRNA expression of P23 in two GC cell lines (AGS and SGC7901) after being treated with P23 shRNA lentivirus. (C) The protein expression of P23, HSP90 and BRCA1 in two GC cell lines after being treated with shP23‐1, shP23‐2 and shP23‐3. (D‐F) BALB/C nude mice are implanted with shNC or shP23‐3 GC cells subcutaneously. (D) The images of subcutaneous tumour nodules formed in two groups of mice. (E) Representative images of P23, HSP90 and BRCA1 in CDX tumour tissue as detected via immunohistochemistry. Scale bars, 50 μm. (F) Data were derived from experiments conducted in triplicate in (E).

Article Snippet: BRCA1 (1:100, Proteintech, 22362‐1‐AP).

Techniques: Western Blot, Expressing, Concentration Assay, shRNA, Immunohistochemistry, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

doi: 10.1007/s12035-025-05423-1

Figure Lengend Snippet: Oxymatrine ameliorates excessive autophagy induced by cerebral I/R in mice. A Western blotting results. B – G Western blotting analyses of LC3-II/I, PINK1, Parkin, NBR1, Beclin-1 and P62 in different groups when normalized to GAPDH. H – K Representative images showing double immunofluorescence staining for autophagy-related proteins (LC3, PINK1, Beclin1, NBR1) co-localized with neurons in different groups. Scale bar = 20 μm. # p < 0.05 vs. Sham, * p < 0.05 vs. I/R

Article Snippet: Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China).

Techniques: Western Blot, Double Immunofluorescence Staining

Journal: Molecular Neurobiology

Article Title: Oxymatrine Alleviates Cerebral Ischemia/Reperfusion Injury By Targeting HDAC1 to Regulate Mitochondria-Related Autophagy and Oxidative Stress

doi: 10.1007/s12035-025-05423-1

Figure Lengend Snippet: Oxymatrine restores mitochondrial homeostasis via coordinated modulation of fission/fusion balance and autophagy in glutamate-treated HT22 cells. A Western blotting for Fis1 and Mfn2. B , C Quantification of western blotting. D Immunofluorescence analysis showing the expression of LC3, PINK1, Beclin-1, NBR1 and P62. Scale bar = 20 μm. E Western blotting for LC3-II/I, PINK1, Beclin-1, NBR1 and P62. F – K Quantification of western blotting. # p < 0.05 vs. Control, * p < 0.05 vs. Glu

Article Snippet: Protein extracts were separated on 10%–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and probed with the following primary antibodies: Apaf-1 (1:1000, ABclonal, Wuhan, China), cleaved-caspase3 (1:500, Proteintech, Wuhan, China), Fis1 (1:2000, Proteintech, Wuhan, China), NBR1 (1:1000, Proteintech, Wuhan, China), Mfn2 (1:4000, Proteintech, Wuhan, China), LC3 (1:1000, ABclonal, Wuhan, China), HDAC1(1:2000, ABclonal, Wuhan, China), PINK1 (1:800, ABclonal, Wuhan, China), Parkin (1:2000, Proteintech, Wuhan, China), Beclin1 (1:2000, Proteintech, Wuhan, China), p62 (1:2000,Proteintech, Wuhan, China).

Techniques: Western Blot, Immunofluorescence, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 1. Rescue of BRCA1 inhibition of ER- transcriptional activity by the HPV E7 and E6 and by other DNA tumor viral oncoproteins. A, domain structure of the DNA tumor virus oncoproteins studied. B, rescue of wtBRCA1 repression of ER- activity by HPV-E7 and HPV-E6 oncogenes. C, rescue of wtBRCA1 repression of ER- activity by adenovirus E1A oncogene. D, rescue of wtBRCA1 repression of ER- activity by the SV40 large T oncogene. E, wtBRCA1 only minimally represses ER- activity in human cervical cancer cell lines. For methodology, subconfluent proliferating cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid (ERE-TK-Luc)(0.25gDNAforeachplasmid)usingLipofectamineTM,asdescribedunder“MaterialsandMethods.”Thecellswerewashed,incubatedwithoutorwith17-estradiol(E2, 10 nM or 1 M) for t 24 h, and harvested for luciferase assays. Luciferase activity was expressed relative to the negative control (0 E2) (B–D) or as a percentage of the E2 positive control (E). The values are expressed as means S.E. of four replicate wells. Each experiment was performed at least twice to ensure reproducibility of the findings. Abbreviations and acronyms are as follows: CDK2, cyclin-dependent kinase 2; CR1/2, conserved region 1/2; CtBP, C-terminal binding protein; C-TERM, C-terminal domain; CXXC, one-half of a zinc coordination motif; cycA, cyclin A; ETQL, PDZ family protein-binding motif; hDLG, homolog of discs large, Drosophila; hScrib, homolog of Scribble, Drosophila; HR, host range domain; Hsc70, heat shock cognate protein 70 kDa; LXCXE, consensus retinoblastoma family protein-binding motif; MAGI-1, membrane-associated guanylate kinase inverted-1; MUPP-1, multiple PDZ domain protein; mut-SV40T, mutant SV40 large T oncogene with defective for RB-binding; NLS, nuclear localization signal; N-TERM, N-terminal domain; PCAF, p300/ CBP-associated factor; PLDLS, CtBP-binding motif; RNA pol II, RNA polymerase II; SV40T, simian virus 40 large T oncogene; TBP, TATA box-binding protein; TEF-1, transcriptional enhancer factor-1; TFIIB, transcription factor IIB; wt, wild-type.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Inhibition, Activity Assay, Virus, Transfection, Expressing, Plasmid Preparation, Luciferase, Negative Control, Positive Control, Binding Assay, Protein Binding, Membrane, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 2. Capture of C-terminally truncated BRCA1 proteins by full-length HPV-E7 and HPV-E6. A, domain structure of BRCA1 protein fragments. B, GST Western blot showing the expression of GST-E7 and GST-E6. The GST-E7 and GST-E6 fusion proteins were visualized by Western blotting using an antibody directed against GST. For reference, 100 l of GST-E7 or GST-E6 was used in the capture assays. C, expression of IVT BRCA1 proteins. The SDS-PAGE autoradiograph shows 10% of the input of each BRCA1 protein used for the GST capture assays. C and E, the arrows indicate the correct bands. D, capture of IVT full-length BRCA1 by GST-E7 and GST-E6. The binding of full-length BRCA1 (amino acids 1–1863) to beadscoatedwithGST-E7orGST-E6wasdeterminedbySDS-PAGEautoradiography.E,captureofsmallerBRCA1proteinfragmentsbyGST-E7andGST-E6.ThebindingofC-truncated BRCA1 proteins to beads coated with GST-E7 or GST-E6 was visualized by SDS-PAGE autoradiography. The abbreviations used are as follows: AD1, activation domain 1; BACH1, BRCA1-associated C-terminal helicase 1; BARD1, BRCA1-associated RING domain 1 protein; BRCT1/2, BRCA1 C-terminal domain 1/2; CtIP, CtBP-interacting protein; HDAC1/2, histone deacetylase 1/2; LMO4, LIM domain only protein 4; NES, nuclear export signal; NLS1, nuclear localization sequence 1; RHA, RNA helicase A; RbAp46/38, retinoblastoma-associated protein 46/48 kDa; TAD, transcriptional activation domain.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Capture-C, Western Blot, Expressing, SDS Page, Autoradiography, Binding Assay, Activation Assay, Histone Deacetylase Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 3. Capture of different IVT BRCA1 protein fragments by full-length GST-E7 and GST-E6. A, domain structure of BRCA1 protein and protein fragments. B, capture of different IVT BRCA1 proteins by full-length GST-E7 protein. The ability of GST-E7 to capture a set of IVT BRCA1 protein fragments spanning the length of the BRCA1 protein was determined. The input lane represents 10% of the IVT protein used in the GST capture assays. C, capture of different IVT BRCA1 proteins by full-length GST-E6 protein. Assays were performed as described in A, except that GST-E6 was used as the bait. D, capture of IVT N-terminal BRCA1 proteins by full-length GST-E7 protein. A group of N-terminal BRCA1 protein fragments was tested for capture by full-length GST-E7 protein. E, capture of IVT N-terminal BRCA1 proteins by full-length GST-E6 protein. A group of N-terminal BRCA1 protein fragments was tested for capture by full-length GST-E6 protein. F, capture of IVT BRCA1 BRCT1 by full-length GST-E7 and GST-E6 proteins. An IVT BRCA1 protein fragment containing the first BRCT1 was tested for capture by the GST-E7 and GST-E6 proteins. NES, nuclear export signal; AD1, activation domain 1.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Activation Assay

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 4. Capture of IVT full-length E7 and E6 proteins by various GST-BRCA1 pro- tein. A, capture of full-length IVT E7 by different GST-BRCA1 proteins. The ability of a set of overlapping GST-BRCA1 proteins to capture in vitro translated full-length E7 was tested. B, capture of full-length IVT E6 proteins by different GST-BRCA1 proteins. The ability of a set of overlapping GST-BRCA1 proteins to capture in vitro translated full- length E7 was tested. C, Western blot showing the expression of different GST-BRCA1 proteins.ThedifferentGST-BRCA1proteinsutilizedinAandBwerevisualizedbyWestern blottingusinganantibodydirectedagainstGST.Thecorrectbandisindicatedbyaboxin the figure.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: In Vitro, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 5. In vivo association of BRCA1 and the E6 and E7 proteins. A, IP of endoge- nous E7 co-precipitates E7 and BRCA1 in SiHa cells. Subconfluent proliferating SiHa human cervical cancer cells were subjected to IP using an anti-E7 antibody and Western- blotted to detect E7 and BRCA1. See “Materials and Methods” for details. B, IP of endog- enous BRCA1 co-precipitates BRCA1 and E7 in SiHa cells. Subconfluent proliferating SiHa cells were subject to IP using an anti-BRCA1 antibody and Western-blotted to detect BRCA1 and E7. See “Materials and Methods” for details. C, IP of BRCA1 co-precipitates BRCA1 and exogenous FLAG-tagged E6 in 293T cells. Subconfluent proliferating 293T cells were transfected overnight with FLAG-E6 and wtBRCA1 expression vectors (10 g ofeachvectorper100-mmdish)usingLipofectamineTM,post-incubatedfor24htoallow gene expression, immunoprecipitated using an anti-BRCA1 antibody, and Western-blot- ted to detect the BRCA1 and FLAG-E6 proteins. D, anti-FLAG IP co-precipitates exoge- nous FLAG-E6 and BRCA1 in 293T cells. 293T cells were transfected as described above in C, post-incubated for 24 h to allow gene expression, immunoprecipitated using an anti- FLAG antibody, and Western-blotted to detect the FLAG-E6 and BRCA1 proteins.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: In Vivo, Western Blot, Transfection, Expressing, Gene Expression, Immunoprecipitation, Incubation

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 6. Capture of BRCA1 by truncated and mutant GST-E7 and E6 proteins. A, schematic diagrams of the truncated/mutant E7 proteins used in this study. B, Western blot showing the expression of truncated and mutant GST-E7 proteins. A set of truncated and mutant GST-E7 proteins were visualized by Western blotting, using an anti-GST antibody. C,captureofIVTBRCA1-(1–302)bytruncatedandmutantGST-E7proteins.DifferentGST-E7proteinsshowninAweretestedfortheirabilitytocaptureaninvitrotranslatedN-terminal portion of BRCA1 (amino acids 1–302). D, capture of IVT BRCA1-(1–771) by truncated and mutant GST-E7 proteins. Different GST-E7 proteins from A were tested for their ability to capture an invitrotranslatedN-terminalportionofBRCA1correspondingtoaminoacids1–771.E,schematicdiagramsofthetruncated/mutantE6proteinsusedinthisstudy.F,Western blot showing the expression of truncated and mutant GST-E6 proteins. A set of truncated and mutant GST-E6 proteins were visualized by Western blotting, using an anti-GST antibody. G, capture of IVT BRCA1-(1–302) by truncated and mutant GST-E6 proteins. The various GST-E6 proteins shown in D were tested for their ability to capture IVT BRCA1-(1–302).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Mutagenesis, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 7.RescueofBRCA1repressionofER-activitybywild-typeversusmutantE7andE6.A,rescueofBRCA1repressionofER-bymutantversuswild-typeE7andE6proteins. Subconfluent proliferating MCF-7 cells in 24-well dishes were transfected overnight with the indicated expression vectors and an estrogen-responsive reporter plasmid (ERE-TK-Luc) (0.25 g DNA for each plasmid) using LipofectamineTM. The cells were washed, incubated without or with 17-estradiol (E2, 10 nM) for t 24 h, and harvested for luciferase assays. Luciferase activity was expressed as a percentage of the E2-positive control; and the values are expressed as means S.E. of four replicate wells. Each experiment was performed at least twice to ensure reproducibility of the findings. B and C, dose responses for the rescue of BRCA1 repression of ER- activity by the wild-type E7 (B) and E6 (C) expression vectors. AssayswerecarriedoutinMCF-7andT47DcellsasdescribedaboveinA,exceptusingdifferentquantitiesoftheE7andE6expressionvectors.Thetotalcontentoftransfectedplasmid DNA was kept constant by the addition of the appropriate quantity of control plasmid.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Positive Control, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 8. Effect of HPV oncoproteins E7 and E6 on other functional activities of BRCA1. A, ability of E7 and E6 to rescue the BRCA1 inhibition of E-box-Luc reporter activity. Subconfluent proliferating MCF-7 or T47D cells in 24-well dishes were trans- fected overnight with the indicated expression vectors and a reporter driven by the c-Myc E-box upstream of a minimal promoter and the luciferase gene (E-box-Luc) (see Ref. 29). After transfection, the cells were washed, post-incubated for 24 h to allow gene expression, and harvested for luciferase assays. Luciferase activity was expressed as a percentage of the positive control (E-box-Luc only, no wtBRCA1, E7, or E6); and the values are expressed as means S.E. of four replicate wells. B, ability of E7 and E6 to rescue the BRCA1 repression of hTERT promoter activity. Assays were performed as described in A, except that a reporter containing the human TERT core promoter upstream of the luciferase gene (29) was utilized instead of the E-box-Luc reporter. Lucif- erase activity was expressed as a percentage of the positive control (hTERT-Luc only, no wtBRCA1, E7, or E6), and the values are expressed as means S.E. of four replicate wells.

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Functional Assay, Inhibition, Activity Assay, Expressing, Luciferase, Transfection, Incubation, Gene Expression, Positive Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 9. Effects of the E6 and E7 oncoproteins on BRCA1, p53, and RB1 protein levels.SubconfluentproliferatingMCF-7orT47Dcellsweretransientlytransfectedover- night with empty pcDNA3 vector, with FLAG wild-type E6 or zinc finger defective mutant E6 (E6-(C66,136G)) (A), or with FLAG wtE7 or zinc finger defective mutant E7 (E7-(C91G)) (B) expression vectors. The cells were washed, post-incubated for 24 h to allow gene expression,andharvestedforWesternblottingtodetecttheE6andE7oncoproteins,the tumor suppressors BRCA1, p53, and RB1, and -actin (control for loading and transfer).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Plasmid Preparation, Mutagenesis, Expressing, Incubation, Gene Expression, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Interaction with Human Papillomavirus Oncoproteins

doi: 10.1074/jbc.m505124200

Figure Lengend Snippet: FIGURE 11. Effect of knockdown of BRCA1 on the ability of the E6 and E7 oncopro- teins to stimulate hTERT promoter activity. A, hTERT promoter assays. Subconfluent proliferating MCF-7 or T47D cells in 24-well dishes were preincubated for 48 h with a previously validated BRCA1 siRNA (29) or the control siRNA (50 nM); transfected over- night with wtE6, wtE7, or pcDNA3 vector; and post-incubated for 24 h to allow gene expression. Luciferase activity was expressed as a percentage of the positive control (hTERT-Luc only, no siRNA, E6, or E7). The values shown are means S.E. of four replicate wells. The data are shown on a linear scale to more easily delineate 2–3-fold changes in reporter gene activity. B, effect of siRNAs on BRCA1 protein levels. Subconfluent prolif- erating MCF-7 cells were treated with no siRNA (vehicle only), BRCA1 siRNA, or control siRNA (50 nM) for 72 h and harvested for Western blotting to detect BRCA1 or -actin (control for loading and transfer).

Article Snippet: The IPs (see above) were electrophoresed on a 4–12% SDS-polyacrylamide gradient gel, transferred to nitrocellulosemembranes (Millipore), and blotted using primary antibodies directed against BRCA1 (C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200); E7 (TVG710Y, Santa Cruz Biotechnology), or the FLAG epitope (M2, mouse monoclonal antibody, Sigma, 1:500 dilution).

Techniques: Knockdown, Activity Assay, Control, Transfection, Plasmid Preparation, Incubation, Gene Expression, Luciferase, Positive Control, Western Blot